ERROR when running dnbc4tools rna

[kobeho24]

Hi there, I am trying to run the v3.0 demo data online https://db.cngb.org/search/sample/CNS1063416/. Errors showed up when running the dnbc4tools rna run using singularity during the count process. The error message is as follows.

2024-05-08 02:33:38,378 - INFO - Merge beads within the same droplet based on correlation values. 2024-05-08 02:33:38,378 - INFO - 2024-05-08 02:33:45,405 - INFO - Generate beads and merge corresponding cell id files /data1/mgi_10x_scrna_comparison/mgi/mouse_brain_1/02.count/barcodeTranslate.txt. 2024-05-08 02:33:45,405 - INFO - 2024-05-08 02:34:06,890 - count - INFO - Executing command: /opt/conda/envs/dnbc4tools/lib/python3.8/site-packages/dnbc4tools/software/tagAdd -n 32 -bam /data1/mgi_10x_scrna_comparison/mgi/mouse_brain_1/01.data/final_sorted.bam -file /data1/mgi_10x_scrna_comparison/mgi/mouse_brain_1/02.count/ba rcodeTranslate.hex.txt -out /data1/mgi_10x_scrna_comparison/mgi/mouse_brain_1/02.count/anno_decon_sorted.bam -tag_check CB:Z: -tag_add DB:Z: 2024-05-08 02:44:28,883 - count - INFO - Program costs: 622s input bam: /data1/mgi_10x_scrna_comparison/mgi/mouse_brain_1/01.data/final_sorted.bam file: /data1/mgi_10x_scrna_comparison/mgi/mouse_brain_1/02.count/barcodeTranslate.hex.txt output bam: /data1/mgi_10x_scrna_comparison/mgi/mouse_brain_1/02.count/anno_decon_sorted.bam tag check: CB:Z: tag add: DB:Z: nthreds: 32

2024-05-08 02:44:28,884 - count - INFO - Executing command: /opt/conda/envs/dnbc4tools/bin/samtools index -@ 32 /data1/mgi_10x_scrna_comparison/mgi/mouse_brain_1/02.count/anno_decon_sorted.bam 2024-05-08 02:45:08,781 - count - INFO - 2024-05-08 02:45:08,782 - count - INFO - Executing command: /opt/conda/envs/dnbc4tools/lib/python3.8/site-packages/dnbc4tools/software/PISA count -one-hit -@ 32 -cb DB -anno-tag GN -umi UB -list /data1/mgi_10x_scrna_comparison/mgi/mouse_brain_1/02.count/cell.id -outdir /data1/mgi_10x_scrna_comp arison/mgi/mouse_brain_1/02.count/raw_matrix /data1/mgi_10x_scrna_comparison/mgi/mouse_brain_1/02.count/anno_decon_sorted.bam 2024-05-08 02:45:08,798 - count - ERROR - Command failed with exit code 127 2024-05-08 02:45:08,798 - count - ERROR - /opt/conda/envs/dnbc4tools/lib/python3.8/site-packages/dnbc4tools/software/PISA: error while loading shared libraries: libcurl.so.4: cannot open shared object file: No such file or directory

What's the solution for this?

Best! Gary

[lishuangshuang0616]

docker or singularity version？

[kobeho24]

singularity version 3.4.1-4.2.ohpc.1.3.9

[lishuangshuang0616]

try

singularity shell dnbc4tools.sif
/opt/conda/envs/dnbc4tools/lib/python3.8/site-packages/dnbc4tools/software/PISA count -h

[kobeho24]

Same error showed up Singularity dnbc4tools.sif:~/tools> /opt/conda/envs/dnbc4tools/lib/python3.8/site-packages/dnbc4tools/software/PISA count -h /opt/conda/envs/dnbc4tools/lib/python3.8/site-packages/dnbc4tools/software/PISA: error while loading shared libraries: libcurl.so.4: cannot open shared object file: No such file or directory

[lishuangshuang0616]

Can you see this libcurl.so.4 in the /opt/conda/envs/dnbc4tools/lib/

[kobeho24]

Singularity dnbc4tools.sif:/opt/conda/envs/dnbc4tools/lib> ls libcurl* libcurl.so libcurl.so.4 libcurl.so.4.8.0

[kobeho24]

singularity exec dnbc4tools.sif dnbc4tools --version 2.1.2

[lishuangshuang0616]

your ~/.bashrc have export LD_LIBRARY_PATH ? If there is, comment it out and then open a new window to run the singularity PISA count -h command.

[kobeho24]

No, there is not.

.bashrc

Source global definitions

if [ -f /etc/bashrc ]; then . /etc/bashrc fi

Uncomment the following line if you don't like systemctl's auto-paging feature:

export SYSTEMD_PAGER=

User specific aliases and functions

export PATH=$HOME/cellranger-7.2.0:$PATH export PATH=$HOME/cellranger-dna-1.1.0:$PATH

>>> conda initialize >>>

!! Contents within this block are managed by 'conda init' !!

conda_setup="$('/data1/miniconda3/bin/conda' 'shell.bash' 'hook' 2> /dev/null)" if [ $? -eq 0 ]; then eval "$__conda_setup" else if [ -f "/data1/miniconda3/etc/profile.d/conda.sh" ]; then . "/data1/miniconda3/etc/profile.d/conda.sh" else export PATH="/data1/miniconda3/bin:$PATH" fi fi unset conda_setup

<<< conda initialize <<<

[kobeho24]

And I run singularity using slurm, would that be an issue? But the same error showed up in singularity shell.

[lishuangshuang0616]

I think it's not related. I need time to test it.

[lishuangshuang0616]

Or you can use the conda version first. I haven't found this problem with my singularity, so it may take more time to find the problem.

[kobeho24]

the conda version works without error